The “proteome” is the complete set of proteins expressed by a cell or population of cells. “Proteomics” is the study of proteomes. An essential step in proteomics is the isolation and identification of the proteins that are being expressed at a particular time, preferably over a range of conditions such as different ages; disease states; and differing exposures to environmental factors such as temperatures, nutrient levels, pharmaceuticals, and other chemical compounds. Quantitative comparisons of protein concentrations under different conditions yield important insights about the health of the organism. For example, there are “marker proteins” whose concentrations change during the progression of disease. Marker proteins have been identified for ailments including many cancers, Alzheimer's disease, schizophrenia, and Parkinson's disease. The accurate measurement of these markers is becoming increasingly important in clinical assays for human disorders and diseases, and can lead to treatments and even cures for a range of maladies.
There are several methods in common use for analyzing complex protein mixtures such as a proteome. In a common strategy the protein mixture is fractionated into individual units, typically using two-dimensional (2D) gel electrophoresis. Proteins are first separated in one dimension, usually by isoelectric focusing (IEF) along a gradient of electrical potential. Each protein's electric charge, which is a function of its constituent amino acids, post-translational phosphorylation, glycosylation, etc., drives the protein along the potential gradient until it reaches its uncharged isoelectric point (pI). The one-dimensional (1D), pI-focused linear array of proteins is then subjected to gel electrophoresis by applying a large electric field in a perpendicular direction. The proteins are driven electrophoretically by the electric field, and separate according to their electrophoretic mobilities, which in turn are determined principally by a protein's size.
Although 2-D gel electrophoresis can resolve more than 1,000 proteins in a single analysis, it has significant limitations. First, its resolving power may be orders of magnitude too small for proteomics, because the sheer number of distinct proteins present in a sample could be on the order of 1,000,000. (The human proteome has been estimated to contain 1.5 million distinct proteins.) Further complexity arises from the numerous interactions that occur among proteins, and between proteins and other ligands. Additionally, proteins are often modified by reactions such as phosphorylation, glycosylation, carbamylation, deamidation, and truncation.
Current bench top electrophoresis methods can be subject to run-to-run reproducibility problems, which can make it difficult to resolve or compare differences between two different gels, whether or the same or different specimens. The ability to make accurate comparisons is particularly important when comparing diseased and healthy specimens, where differences can be subtle; or when comparing the proteome of an organism when it is subjected to different environments or drug exposures.
A typical analysis involves several time-consuming, labor-intensive, skilled operations. For example, 2D gel electrophoresis typically requires several days of development time, followed by staining the gel to visualize proteins, picking “protein spots” from the gel for enzymatic digestion, separating the resulting peptide fragments, and mass spectrometry (MS) to identify those fragments.
Another challenge in protein separation is that the concentrations of different proteins can vary enormously, sometimes by up to ten orders of magnitude. An important consequence of the wide concentration range is that different stains are typically used to visualize spots within a gel plate. The stains have differing sensitivities to protein structures and concentrations, and are not reliably quantitative. Stain variability may affect subsequent analysis, e.g., an excised, stained gel spot subjected to mass spectrometry may yield different results depending upon the nature of the stain-protein interaction, which may be irreproducible from gel-to-gel.
Because of the difficulties and limitations in analyzing proteins by conventional, bench-top processing techniques for proteomic studies, there is an unfilled need for alternative techniques to analyze proteins, mixtures of proteins, and proteomes.
The peak capacity, P, refers to the maximum number of components that can be resolved in any one separation. A requirement of any successful multi-dimensional procedure is orthogonality, which means that the selected single dimensions possess different but compatible separation mechanisms. Furthermore, any separation step in a series of separations should not un-do any separations that were achieved by prior steps. When separation modes are truly orthogonal, the peak capacity P of a multi-dimensional separation is the product of the n peak capacities of its constituent 1D methods, P=P1×P2× . . . Pn-1×Pn. Complete orthogonality is rarely obtained with any multi-dimensional separation technique; i.e., the peak capacity found in practice is generally lower than the theoretical maximum.
Microfluidic devices have been used to implement various separation techniques. Typically, microfluidic devices apply an electric field to induce or to switch fluid flow. To achieve reproducible, high-resolution separations, it should be possible to controllably inject a fluid sample “plug,” a predetermined volume of fluid sample, into a separation conduit. For fluid samples containing high molecular weight, charged biomolecules such as proteins, microfluidic devices containing an electrophoresis separation channel a few millimeters to a few centimeters long may be used to separate small fluid samples having a plug length on the order of a few micrometers. High-sensitivity detection techniques such as laser-induced fluorescence (LIF) may be used to monitor separated sample components. The analyte stream may also be fed into other analytical devices such as a mass spectrometer for detailed characterizations. Microfluidic devices have been reported for 2D separations using IEF and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic peptide separations, solid-phase digestion using enzymatic bioreactors, and interfacing to either matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) mass spectrometry.
Recently, on-line 2D capillary electrophoresis (CE) has been reported, with high separation efficiencies, good resolution and convenient coupling to MS. Other 2D CE techniques have included coupling of isotachophoresis (ITP) and capillary zone electrophoresis (CZE), IEF, and ITP, CZE and capillary gel electrophoresis (CGE), capillary sieving electrophoresis (CSE) and micellar electrokinetic chromatography (MEKC), MEKC and IEF, IEF and CGE, IEF and CSE, and CE and CZE. These 2D CE separations typically have peak capacities in the range of 500-1,000.
Microchip capillary electrophoresis (μ-CE) represents a promising avenue for proteome analysis, offering advantages such as reduced consumption of sample and reagents, shorter analysis times, low “dead” volumes when multiple separation techniques or “dimensions” are coupled, and the ability to integrate complex geometries into a small area. In addition, microchips may be fabricated in a variety of polymer substrates suitable for different separations, using a single replication master. Journal of Chromatography A 2006, 1111 238-251.
However, only a few successful instances of 2D μCE separations have previously been reported. On-chip 2D CE separations were described by Ramsey et al. for peptides using fluorescence detection on a glass microchip. Analytical Chemistry 2003, 75, 3758-3764; Analytical Chemistry 2001, 73, 2669-2674; Analytical Chemistry 2000, 72, 5244-5249. The proteins were digested into peptides off-chip prior to conducting the separation. 2D μ-CE separations of proteins without prior digestion have been reported by coupling IEF with CZE, or IEF with CGE. Analytical Chemistry 2003, 75, 1180-1187; Analytical Chemistry 2004, 76, 4426-4431; Lab on a Chip 2004, 4, 18-23; Analytical Chemistry 2004, 76, 4426-4431; Analytical Chemistry 2004, 76, 742-748; Analytical Chemistry 2004, 76, 1359-1365; Analytical Chemistry 2002, 74, 1772-1778; Journal of Chromatography, A 2005, 1087, 177-182; Electrophoresis 2007, 28, 422-428.
Most prior 2D separations of proteins on microchips have used IEF as one of the separation dimensions. However, IEF is generally incompatible with fluorescence labeling, because the incorporation of a fluorescent tag will generally alter a molecule's isoelectric point. Furthermore, diffusion between focused bands following IEF decreases the efficiency and resolution following transfer into the second dimension. Some investigators have reported low reproducibility in the IEF dimension.
A capillary electrochromatography device can be made by placing a high surface-area stationary or pseudo-stationary phase in an electrophoresis microchannel. Electroosmotic flow (EOF) drives the mobile liquid phase through the channel, and analyte-stationary phase interactions lead to different analyte retention times in the microchannel. A modification is to make the high surface-area stationary phase reactive, e.g., by attaching an enzyme or other catalyst to digest larger protein molecules into smaller, more easily identifiable polypeptides.
H. Shadpour et al., “Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips,” Analytical Chemistry, 2006, 78, 3519-3527 discloses 2D electrophoretic separations of proteins in a poly(methyl methacrylate)-(PMMA-) based microchip. Sodium dodecyl sulfate microcapillary gel electrophoresis (SDS μ-CGE) and MEKC were used as the separation modes. The microchip was prepared by hot embossing into PMMA from a brass mold master fabricated via high-precision micromilling. The microchip incorporated a 30-mm SDS μ-CGE and a 10-mm MEKC dimension.
G. Chen et al., “Functional template-derived poly(methyl methacrylate) nanopillars for solid-phase biological reactions,” Chemistry of Materials, vol. 19, pp. 3855-3857 (2007) discloses the fabrication of ultra-high aspect ratio polymer nanopillars by a template-synthesized approach, and their use as high surface area scaffolds for attaching biomolecules. See also G. Chen et al., “Free-standing, erect ultra-high-aspect-ratio polymer nanopillar and nanotube ensembles,” Langmuir (accepted for publication, 2007).
U.S. patent application publication 2003/0089605 discloses a microfluidic system and method to analyze large numbers of compounds, using an upstream separation module (such as a multidimensional separation device), a microfluidic device for on-device protein digestion of substantially separated proteins received from the upstream separation module, a downstream separation module for separating digestion products of the proteins, a peptide analysis module, and a processor for determining the amino acid sequence of the proteins.
N. Kaji et al., “Separation of long DNA molecules by quartz nanopillar chips under a direct current electric field,” Anal. Chem., vol. 76, pp. 15-22 (2004) discloses the production of SiO2 nanopillars by a dry etching process. The nanopillars were employed in a channel to separate DNA fragments. The nanopillars were reported to have a diameter 100-500 nm, and a height 500-5000 nm.
M. Slovakova et al., “Use of self assembled magnetic beads for on-chip protein digestion,” Lab Chip, vol. 5, pp. 935-942 (2005) discloses the use of grafted trypsin magnetic beads for protein digestion in a PDMS microchannel.
J. Duan et al., “Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry,” J. Chrom. A, vol. 1106, pp. 165-174 (2006) discloses an enzymatic microreactor prepared in a fused-silica capillary by in situ polymerization of acrylamide, N-acryloxysuccinimide, and ethylene dimethacrylate in the presence of a binary porogenic mixture of dodecanol and cyclohexanol; and the use of the microreactor for the digestion of cytochrome c over immobilized trypsin.
R. Foote et al., “Preconcentration of proteins on microfluidic devices using porous silica membranes,” Anal. Chem., vol. 77, pp. 57-63 (2005) discloses the electrophoretic concentration of proteins on a microfabricated device using a porous silica membrane between microchannels, followed by separation in a coated or uncoated channel.
J. Liu et al., “Surface-modified poly(methyl methacrylate) capillary electrophoresis microchips for protein and peptide analysis,” Anal. Chem., vol. 76, pp. 6948-6955 (2004) discloses the use of surface-modified PMMA for capillary electrophoresis.
There is a continuing, unfilled need for high-surface-area solid supports for protein digestion, analysis, and other uses. Supports that have been used include gel pads, beads, monoliths, and lithographically fabricated micro- and nanopillars. Nanopillars in particular have the potential to be useful. Their height, aspect ratio, and spacing can be selected to match the biological reagents, diffusion attributes, and other factors to the particular need. However, prior methods for producing nanopillars are expensive and have low throughput. Templates have been used to form polymers into ordered pillar arrays, but this technique has not successfully produced small-diameter nanopillars (diameter less than about 500 nm) with the aspect ratios higher than about 2-5; and the ability to withstand the solution processing conditions required to attach chemical or biological compounds onto them.